Increase In Membrane Viscosity Research Articles

Effects of 2,3-diphosphoglycerate on the mechanical properties of erythrocyte membrane

Investigation by Schindler et al and Sheetz and Casaly have indicated that high (approximately 10 mmol/L) concentrations of 2,3- diphosphoglycerate (2,3-DPG) have a destabilizing effect on erythrocyte membrane and the membrane skeleton. We have investigated changes in the membrane mechanical properties that occur at elevated 2,3-DPG levels in both intact cells and ghosts. The membrane shear modulus, viscoelastic recovery time constant, critical force, “plastic” viscosity, and material relaxation time constant were measured by standard micropipette and flow channel techniques. Intact cells showed no change in properties at physiologic ionic strength and 2,3-DPG concentrations of about 20 mmol/L, except for an increase in membrane viscosity resulting from an increased cellular hemoglobin concentration that occurs when the 2,3-DPG concentration is elevated. At ionic strengths 20% below physiologic and 2,3-DPG concentrations of approximately 20 mmol/L, decreases in membrane shear modulus and membrane viscosity were observed. In ghosts, no changes in these properties were observed at a 2,3-DPG concentration of 10 mmol/L and ionic strengths as low as 25% below physiologic, but a decrease in the force required to form tethers (critical force) was observed at physiologic ionic strength. The decrease in membrane shear modulus and viscosity of intact cells and the reduced critical force in ghosts are consistent with the results of other investigators. However, the difference in the effects of 2,3-DPG on ghosts and intact cells indicates that the effects of 2,3-DPG depend strongly on the conditions of the experiment. It appears unlikely that 2,3-DPG affects erythrocyte membrane material properties under physiologic conditions.

Effects of 2,3-Diphosphoglycerate on the Mechanical Properties of Erythrocyte Membrane

Investigation by Schindler et al and Sheetz and Casaly have indicated that high (approximately 10 mmol/L) concentrations of 2,3-diphosphoglycerate (2,3-DPG) have a destabilizing effect on erythrocyte membrane and the membrane skeleton. We have investigated changes in the membrane mechanical properties that occur at elevated 2,3-DPG levels in both intact cells and ghosts. The membrane shear modulus, viscoelastic recovery time constant, critical force, "plastic" viscosity, and material relaxation time constant were measured by standard micropipette and flow channel techniques. Intact cells showed no change in properties at physiologic ionic strength and 2,3-DPG concentrations of about 20 mmol/L, except for an increase in membrane viscosity resulting from an increased cellular hemoglobin concentration that occurs when the 2,3-DPG concentration is elevated. At ionic strengths 20% below physiologic and 2,3-DPG concentrations of approximately 20 mmol/L, decreases in membrane shear modulus and membrane viscosity were observed. In ghosts, no changes in these properties were observed at a 2,3-DPG concentration of 10 mmol/L and ionic strengths as low as 25% below physiologic, but a decrease in the force required to form tethers (critical force) was observed at physiologic ionic strength. The decrease in membrane shear modulus and viscosity of intact cells and the reduced critical force in ghosts are consistent with the results of other investigators. However, the difference in the effects of 2,3-DPG on ghosts and intact cells indicates that the effects of 2,3-DPG depend strongly on the conditions of the experiment. It appears unlikely that 2,3-DPG affects erythrocyte membrane material properties under physiologic conditions.

Chilling Sensitivity of Tomato Fruit in Relation to Ripening and Senescence

Abstract Postchilling ion leakage, respiration, and C2H4 biosynthesis were used to measure the degree of chilling injury to fruit of tomato (Lycopersicon esculentum Mill. ‘Heinz 1350’). Chilling sensitivity, as measured by ion leakage, first declined as the tomatoes began to ripen and then increased during the late stages of ripening. Both C2H4 biosynthesis and respiration rate were stimulated during the chilling response early in ripening. In the nonripening mutant, nor, chilling sensitivity did not show the early decline but showed the increase during senescence. Variation in chilling sensitivity during tomato ripening was biphasic, with a decline at the onset of ripening followed by a senescence-related increase. It is proposed that the latter may be due to an increase in membrane viscosity.

The Modulatory Effect of Membrane Viscosity on Structural and Functional Properties of the Anion Exchange Protein of Human Erythrocytes

The sterol content of human erythrocyte membranes was modified by polyvinylpyrrolidone (PVP)-mediated enrichment or depletion of cholesterol (CHL) or incorporation of cholesteryl hemisuccinate (CHS). The effects of these modifications on osmotic fragility and anion exchange protein (AEP) disposition and function were evaluated. CHS enrichment was fast (1 hr, 37 degrees C) and led to a concentration-dependent crenation as well as a decrease in osmotic cell fragility, in parallel with increased membrane microviscosity. CHL caused similar but considerably less marked effects due to slower incorporation rates into membranes. CHS enrichment of cells induced susceptibility of AEP to trypsin, a protease which otherwise does not affect AEP in intact cells. Although transport rates of monosaccharides, nucleosides, and anions were markedly slowed down by CHS enrichment of cells in parallel with increased membrane viscosity, anion transport was the most affected. The temperature profile of anion transport in CHS-enriched cells revealed a 10-kcal/mol increase in the enthalpy of activation relative to normal cells. Anion transport measured in heteroexchange conditions (Cl in--pyruvate out) and (Cl in-sulfate out) was relatively more susceptible to CHS modification than when it was measured in homoexchange conditions (Cl in-Cl out). The results of these measurements indicate that CHS-mediated increase in membrane viscosity affects AEP translocation capacity and transmembrane disposition via changes in lipid compressibility. Specific effects of CHS on AEP function, however, could not be ruled out.

Abnormal erythrocyte fragmentation and membrane deformability in paroxysmal nocturnal hemoglobinuria.

Hemolysis in paroxysmal nocturnal hemoglobinuria (PNH) is considered to be a result of an intrinsic membrane defect. This defect may result in abnormal material properties of PNH erythrocytes. To examine this hypothesis, fragmentation failure, and membrane deformability were assessed in the absence of complement by micropipette techniques. Membrane viscosity was determined by observing relaxation of deformed cells. Results show a bimodal distribution of force for membrane failure, membrane viscoelasticity, and elastic shear modulus. One population requires significantly less force for fragmentation, mean 0.56 X 10(-6) dyne; has increased membrane viscosity, mean 0.205 X 10(-2) dyne sec/cm; and has decreased elastic shear modulus, mean 0.56 X 10(-2) dyne/cm. A second population resembles control with fragmentation force, mean 1.19 X 10(-6) dyne, control 1.05 X 10(-6) dyne; membrane viscosity, mean 0.112 X 10(-2) dyne/cm, control 0.102 X 10(-2) dyne sec/cm; elastic shear modulus, mean 0.70 X 10(-2) dyne/cm, control 0.78 X 10(-2) dyne/cm. The percent of cells with abnormal material properties corresponds to the percent of PNH III cells determined by complement lysis. Thus, the hemolysis attributed to an abnormal clone of erythrocytes in PNH is associated with an intrinsic membrane abnormality which predisposes to lysis.

Lipopolysaccharide interaction with rabbit erythrocyte membranes

In this study we have characterized the association of a polysaccharide-deficient, lipid-rich lipopolysaccharide (LPS) with rabbit erythrocytes (RaRBC). With polymyxin B sulfate-mediated hemolysis as a probe, we have shown that Salmonella minnesota R595 LPS interacts with RaRBC in two distinguishable steps. The first step whereby RaRBC exposed to LPS are rendered sensitive to polymyxin B-initiated lysis probably represents absorption of LPS to the RaRBC membrane. We investigated two possible mechanisms for the subsequent time-dependent decrease in response of LPS-treated RaRBC to polymyxin B. We found that the decay in polymyxin B susceptibility of LPS-treated RaRBC cannot be attributed to a decrease in binding of LPS to the RaRBC. On the other hand, our results are consistent with a time-dependent rearrangement of the amphipathic LPS within the lipid bilayer of the RaRBC membrane. In particular, at lower incubation temperatures of RaRBC and LPS, the decay in polymyxin B-induced hemolysis is slower, presumably, because the increased membrane viscosity allows less rapid rearrangement of LPS within the lipid bilayer. A putative hydrophobic intercalation of LPS into a mammalian cell membrane may be of importance in LPS stimulation of responsive cells.

Protein phosphorylation-induced State I-State II transitions are dependent on thylakoid membrane microviscosity

Incorporation of cholesterol hemisuccinate into thylakoid membranes decreased the membrane fluidity as measured by polarized fluorescence from 1,6-diphenyl-1,3,5-hexatriene. Increasing membrane viscosity in this manner did not inhibit the thylakoid membrane protein kinase. In contrast the effects of the protein phosphorylation on State I-State II transitions, which were observed in untreated membranes, were abolished. This observation is interpreted as indicating that protein phosphorylation-induced energy transfer changes are sensitive to membrane viscosity because they might require a lateral migration of the light-harvesting complex serving Photosystem II from grana to stromal lamellae. Cation effects on room- and low-temperature fluorescence emission properties and membrane adhesion were not abolished in these cholesterol hemisuccinate-treated membranes.

Biochemical changes of rat brain membranes with aging.

Modification of membrane composition and enzymatic activities both in total brain homogenate and purified synaptic plasma membrane of 3 and 24 month old rats has been investigated. Protein, cholesterol and phospholipid content and (Na+, K+)ATPase and 2',3' cyclic nucleotide phosphohydrolase activities were determined. The major changes occurred in the whole homogenate where a general increase in total protein and cholesterol content with age and a significant increase of the cholesterol/phospholipids molar ratio has been detected. In S.P.M. aging process induced a decrease of protein, cholesterol and phospholipids content associated with an increased membrane viscosity and a decrease of delta E. These data are consistent with a change in the structural organization and in the distribution pattern of different cell population in the aging brain. A possible artifactual effect of freezing on the reported parameter is also discussed.

Geometric, Osmotic, and Membrane Mechanical Properties of Density-Separated Human Red Cells

Although there is evidence that the deformability of the entire red blood cell (RBC) decreases during aging, reports on changes in relevant specific properties associated with the aging process are limited and not in total agreement. The purpose of this study was to evaluate some of the factors that might contribute to this decreased deformability. Geometric, osmotic, and membrane mechanical properties of unfractionated, top ("young") and bottom ("old") RBC from 5 healthy adult donors were measured using micropipette techniques. Surface area, volume, and diameter of RBC were measured at osmolalities of 297, 254, 202, and 153 mosm/kg. Two membrane mechanical properties, surface shear modulus of elasticity (mu) and time constant (tc) of viscoelastic recovery, were studied only in isotonic media. At each of the osmolalities, volume and surface area of the bottom cells were about 25% lower than those of the top cells. Bottom cells showed smaller increases in volume with decreasing osmolality than top cells; the surface area remained constant with changing osmolality for all three groups. The surface area-to-volume ratio and the minimum cylindrical diameter of the bottom cells were essentially identical to the top cells. However, both the surface area index (actual are of RBC divided by area of a sphere of same volume) and the swelling index (maximal volume divided by actual volume) of the bottom cells were significantly lower than top RBC. The shear modules of elasticity (mu) was about 0.006 dyne/cm in all 3 RBC populations, indicating that the forces necessary to deform a portion of the membrane did not change with RBC aging. The viscoelastic time constant (tc) was 0.148 +/- 0.020 (SD) sec for the bottom RBC and 0.099 +/- 0.017 sec for the top cells. This difference indicates that shape recovery following membrane deformation is delayed in old RBC. The membrane surface viscosity (eta), calculated as the product of tc times mu was 0.95 +/- 0.22 x 10(-3) dyne-sec/cm for the bottom cells and 0.54 +/- 0.15 x 10(-3) for the top RBC. These data indicate that the relative deficit in membrane surface area and the increased membrane viscosity of old RBC may be important determinants for their decreased deformability and their eventual removal from the circulation.

Geometric, osmotic, and membrane mechanical properties of density- separated human red cells

Although there is evidence that the deformability of the entire red blood cell (RBC) decreases during aging, reports on changes in relevant specific properties associated with the aging process are limited and not in total agreement. The purpose of this study was to evaluate some of the factors that might contribute to this decreased deformability. Geometric, osmotic, and membrane mechanical properties of unfractionated, top (“young”) and bottom (“old”) RBC from 5 healthy adult donors were measured using micropipette techniques. Surface area, volume, and diameter of RBC were measured at osmolalities of 297, 254, 202, and 153 mosm/kg. Two membrane mechanical properties, surface shear modulus of elasticity (mu) and time constant (tc) of viscoelastic recovery, were studied only in isotonic media. At each of the osmolalities, volume and surface area of the bottom cells were about 25% lower than those of the top cells. Bottom cells showed smaller increases in volume with decreasing osmolality than top cells; the surface area remained constant with changing osmolality for all three groups. The surface area-to-volume ratio and the minimum cylindrical diameter of the bottom cells were essentially identical to the top cells. However, both the surface area index (actual are of RBC divided by area of a sphere of same volume) and the swelling index (maximal volume divided by actual volume) of the bottom cells were significantly lower than top RBC. The shear modules of elasticity (mu) was about 0.006 dyne/cm in all 3 RBC populations, indicating that the forces necessary to deform a portion of the membrane did not change with RBC aging. The viscoelastic time constant (tc) was 0.148 +/- 0.020 (SD) sec for the bottom RBC and 0.099 +/- 0.017 sec for the top cells. This difference indicates that shape recovery following membrane deformation is delayed in old RBC. The membrane surface viscosity (eta), calculated as the product of tc times mu was 0.95 +/- 0.22 x 10(-3) dyne-sec/cm for the bottom cells and 0.54 +/- 0.15 x 10(-3) for the top RBC. These data indicate that the relative deficit in membrane surface area and the increased membrane viscosity of old RBC may be important determinants for their decreased deformability and their eventual removal from the circulation.

Positive skin tests with autologous tumor cells of increased membrane viscosity

Skin tests were carried out with irradiated autologous tumor cells in patients with solid tumors (large-bowel carcinoma, gastric carcinoma, malignant melanoma, and breast cancer). With untreated cells, the skin reaction was negative or very weakly positive. A strong skin reaction, however, was elicited with cells of elevated membrane lipid microviscosity. This was induced by incorporation of cholesterol or, more effectively, by cholesterly hemisuccinate (CHS). A fair correlation was observed between the level of skin reaction with CHS-treated cells and the clinical stage of the tumor. It is suggested that rigidification of the cell membrane lipid layer exposes latent tumor-associated antigens, which enhances the cell-specific tumor immunogenicity. CHS-treated cells could therefore be of clinical value in active immunotherapy of human cancer.

877 FETAL RED CELLS (FC) DIFFER IN ELASTICITY, VISCOSITY, AND ADHESION FROM ADULT RED CELLS (AC)

Blood hyperviscosity occurs in 3-5% of full-term infants, usually accompanies central venous hematocrit (HCT)>65%, but may occur at HCT 60-65%. Small differences in red cell deformability have major influence on blood viscosity at high HCT. We studied the whole cell deformability of washed FC and AC by counting the % cells aspirated more than 10μm into 2μm pore diameter nuclepore filters. FC were more deformable with 88±7% having aspiration length >10μm compared to 19±4% for AC (p<.01). More extensible membranes have lower membrane elasticity (μ in 10-3 dyne/cm); μ was calculated from small nipple extensions into 0.6μm pore diameter nuclepore filters. FC had lower μ than AC (μFC=4.39, μAC=8.06; p<.05). Subpopulations of FC separated by density gradient showed invariant μ with increasing MCHC and decreasing cell volume. Work from a collaborating laboratory (Blackshear et al, 1980 Gotenburg Filtration Symposium) has shown increased membrane viscosity (η) of FC (ηFC=6×10-4; ηAC=3×10-4 poise-cm). More FC were adherent to the filter than AC in the special, purged filter chamber (FC 9.0%, AC 0.3%). The altered whole cell deformability and membrane elasticity of FC would ameliorate the flow retardation influences of FC membrane viscosity and adhesion. Evaluation of these rheological properties in newborn hyperviscosity syndrome may determine who would benefit from phlebotomy.

Cell-substratum and cell-monolayer adhesion are dependent upon cellular cholesterol biosynthesis

Incubation of Chinese hamster lung (Dede) cells in medium containing 25-hydroxycholesterol resulted in the suppression of cellular cholesterol biosynthesis and a decrease in the cellular concentration of cholesterol so that the cholesterol/phospholipid molar ratio was reduced to approx. 50% of the control value. When these ‘sterol-depleted’ cells were allowed to attach to a serum-coated glass substratum, an inhibition of the rate and extent of attachment relative to the controls was observed. After 1 h incubation, only 10–25% of the 25-hydroxycholesterol-treated cells were attached, compared with virtually 100% of the control cells. Similar results were obtained with Chinese hamster ovary cells and with mouse L cells. Inhibition of adhesion was dependent upon the dose of—and time of exposure to—the oxygenated sterol. This inhibition was prevented when either cholesterol or mevalonic acid (the product of the enzymatic reaction inhibited by 25-hydroxycholesterol) was added with the 25-hydroxycholesterol, indicating that the effect of the sterol was primarily due to suppression of cholesterol synthesis. In addition, the inhibitor had no effect on the growth of a 25-hydroxycholesterol-resistant mutant or on the rate or extent of its adhesion to the substratum. Other oxygenated sterols (e.g. 22-ketocholesterol, 7β-hydroxycholesterol, 20α-hydryxycholesterol and 5α-cholest-8(14)-en-3β-ol-15-one) effected qualitatively similar inhibitions of the rates of sterol synthesis and cell-substratum attachment. The decreased adhesion observed when cultures were treated with inhibitors of cholesterol synthesis was not alleviated at low temperatures, suggesting that the effects of the inhibitor could not be overcome by a temperature-dependent increase in membrane viscosity. The rate and extent of adhesion of either Chinese hamster lung cells or L cell to several cell monolayers was also reduced in response to sterol depletion. These results suggest that membrane lipids, particularly cholesterol, play a significant role in cell-substratum and cell-cell adhesion.

Elevation of apparent membrane viscosity in ovarian granulosa cells by follicle-stimulating hormone

Continued exposure of cultured granulosa cells to follicle-stimulating hormone (FSH) induced: (i) a rise in apparent membrane microviscosity, as reflected by an increase in fluorescence polarization of the lipid-soluble probe, 1,6-diphenyl-1,3,5,-hexatriene; and (ii) a progressive decline in the cyclic AMP response to renewed challenge with the same hormone. Both changes were reduced or prevented by pretreatment of the cells with oleic or linoleic acid, agents which reduce membrane viscosity, but not by elaidic or palmitic acid which increase the rigidity of membrane lipids. Other agents that inhibited FSH-induced changes in membrane fluidity (gonadotropin-releasing hormone, actinomycin D and cycloheximide) also prevented desensitization to FSH. Cyclic AMP and cyclic GMP derivatives did not mimic the effects of FSH on apparent membrane viscosity or desensitization. Changes in membrane fluidity are unlikely to be the sole cause of desensitization since (i) pretreatment of the cells with fatty acids that increase lipid viscosity did not induce desensitization to FSH, and (ii) desensitization of granulosa cells to lutropin and prostaglandin E 2 by exposure to the homologous hormone was not attended by increased membrane viscosity. The experiments described provide the first example of a hormonally induced increase in the target cell apparent membrane viscosity.